CN109641931A - The synthetic method of ribonucleic acid H- phosphate ester monomer and the oligonucleotide synthesis for having used the monomer - Google Patents

The synthetic method of ribonucleic acid H- phosphate ester monomer and the oligonucleotide synthesis for having used the monomer Download PDF

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Publication number
CN109641931A
CN109641931A CN201780051108.5A CN201780051108A CN109641931A CN 109641931 A CN109641931 A CN 109641931A CN 201780051108 A CN201780051108 A CN 201780051108A CN 109641931 A CN109641931 A CN 109641931A
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unsubstituted
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chemical formula
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CN109641931B (en
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井上聪
姬野康平
南海浩
南海浩一
田中睦生
村上悌
村上悌一
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Gene Design Co Ltd
National Institute of Advanced Industrial Science and Technology AIST
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Gene Design Co Ltd
National Institute of Advanced Industrial Science and Technology AIST
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic System
    • C07F9/02—Phosphorus compounds
    • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
    • C07F9/6558—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing at least two different or differently substituted hetero rings neither condensed among themselves nor condensed with a common carbocyclic ring or ring system
    • C07F9/65586—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing at least two different or differently substituted hetero rings neither condensed among themselves nor condensed with a common carbocyclic ring or ring system at least one of the hetero rings does not contain nitrogen as ring hetero atom
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic System
    • C07F9/02—Phosphorus compounds
    • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
    • C07F9/6561—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing systems of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring or ring system, with or without other non-condensed hetero rings
    • C07F9/65616—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing systems of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring or ring system, with or without other non-condensed hetero rings containing the ring system having three or more than three double bonds between ring members or between ring members and non-ring members, e.g. purine or analogs
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00—Processes for the preparation of sugar derivatives
    • C07H1/02—Phosphorylation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/06—Pyrimidine radicals
    • C07H19/067—Pyrimidine radicals with ribosyl as the saccharide radical
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/16—Purine radicals
    • C07H19/167—Purine radicals with ribosyl as the saccharide radical
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Abstract

The present invention relates to the methods of the synthetic method of ribonucleic acid H- phosphate ester monomer and the oligonucleotide synthesis for having used the monomer.The manufacturing method of 2 ' positions of ribonucleotide monomer needed for synthesizing RNA oligonucleotide the present invention relates to selectively protection and the H- phosphate nucleoside derivates that can be manufactured with low cost.The present invention is characterized in that; it is reacted at low temperature by the hydroxyl of the 2 ' positions and 3 ' positions that keep reactivity slightly different with aromatic series carboxylic acid halides; to carry out the esterification of 2 ' positions selectivity; then by capturing the hydroxyl of 3 ' positions in one pot, the rearrangement of 2 ' positions and 3 ' positions without causing acyl group using phosphono.

Description

The synthetic method of ribonucleic acid H- phosphate ester monomer and the few nucleosides for having used the monomer Acid synthesis
Technical field
The present invention relates to selectively protection synthesis RNA oligonucleotide needed for ribonucleotide monomer 2 ' positions and can The manufacturing method for the H- phosphate nucleoside derivates being manufactured with low cost.
Background technique
Since self-discovery RNAi, RNA oligonucleotide is headed by siRNA it can be expected that in drug and in worldwide It is developed.
Summary of the invention
The solution to the problem
The inventors of the present invention have made intensive studies, and are as a result used as by using phosphite ester for importing manufacture RNA monomer The phosphorus reagent of required phosphorus part so as to easily remove the residue of phosphorus reagent, and develops what operation was significantly improved Synthetic method.
The present invention also provides projects below.
(project 1)
A method of general formula compound represented below or its salt or its solvate are manufactured,
[chemical formula 1]
(in formula (1), R1It indicates selected from the group being made of substituted or unsubstituted aryl and substituted or unsubstituted heteroaryl Group, R2Indicate the protecting group of hydrogen atom or hydroxyl, B indicates nucleic acid base optionally with protecting group)
This method includes process 1 and process 2,
Process 1 is that [chemical formula 2] is made to react the process for obtaining [chemical formula 4] with [chemical formula 3],
[chemical formula 2]
[chemical formula 3]
(in formula (3), X is halogen atom)
[chemical formula 4]
Process 2 is to make [chemical formula 5] and P (OR3)(OR3’) OH react obtain [chemical formula 6] process,
[chemical formula 5]
[chemical formula 6]
(formula P (OR3)(OR3’) in OH, R3And R3’It separately indicates selected from by substituted or unsubstituted aryl and substitution Or the group of the group of unsubstituted heteroaryl composition),
The process 1 and the process 2 carry out in 1 reaction vessel.
(project 2)
According to method described in above-mentioned project, wherein the optionally nucleic acid base with protecting group is selected from by such as the following group At group,
[chemical formula 7]
(in formula (7), R3a、R3bAnd R3cIt separately indicates selected from by hydrogen atom, substituted or unsubstituted straight chain or branch Alkanoyl, substituted or unsubstituted alkyl, substituted or unsubstituted naphthenic base, takes substituted or unsubstituted aryl-acyl The group or R of the group of generation or unsubstituted aryl and substituted or unsubstituted heteroaryl composition3aWith R3bIt is formed together amidine type The group of protecting group).
(project 3)
The method according to any one of above-mentioned project, wherein aforementioned amidine type protecting group is N, N- dimethylformamidinyl Or N, N- dimethyl ethanamidine base.
(project 4)
The method according to any one of above-mentioned project, wherein R1For substituted or unsubstituted aryl.
(project 5)
The method according to any one of above-mentioned project, wherein aforementioned substituted or unsubstituted aryl is phenyl.
(project 6)
The method according to any one of above-mentioned project, wherein the protecting group of aforementioned hydroxy is selected from being protected by ether system The protecting group for the group that base, silyl ether system protecting group, acetal system protecting group and acyl group system protecting group form.
(project 7)
The method according to any one of above-mentioned project, wherein aforementioned ether system protecting group are as follows:
[chemical formula 8]
(in formula (R2a), R4a1、R4a2、R4a3、R4a4、R4a5、R4b1、R4b2、R4b3、R4b4、R4b5、R4c1、R4c2、R4c3、R4c4With R4c5Separately for selected from by hydrogen atom, substituted or unsubstituted straight or branched alkoxyl and substituted or unsubstituted straight The group of the group of chain or branched alkyl composition.).
(project 8)
The method according to any one of above-mentioned project, wherein aforementioned substituted or unsubstituted linear chain or branched chain alcoxyl Base is linear chain or branched chain perfluoro alkoxy.
(project 9)
The method according to any one of above-mentioned project, wherein aforementioned substituted or unsubstituted linear or branched alkyl group For linear chain or branched chain perfluoroalkyl.
(project 10)
The method according to any one of above-mentioned project, wherein aforementioned silyl ether system protecting group are as follows:
[chemical formula 9]
(in formula (R2b), R4d、R4eAnd R4fSeparately for selected from by substituted or unsubstituted linear or branched alkyl group, The group of the group of substituted or unsubstituted aryl and substituted or unsubstituted alkenyl composition.).
(project 11)
The method according to any one of above-mentioned project, wherein aforementioned substituted or unsubstituted alkenyl is allyl.
(project 12)
The method according to any one of above-mentioned project, wherein aforementioned silyl ether system protecting group are as follows:
[chemical formula 10]
(in formula (R2c), R4g、R4hAnd R4iSeparately for selected from not taking by substituted or unsubstituted alkyl, substitution or The group of the group of the silicyl in generation and substituted or unsubstituted silicyl oxygroup alkyl composition.).
(project 13)
The method according to any one of above-mentioned project, wherein aforementioned silicyl is replaced by 1~3 alkyl.
(project 14)
The method according to any one of above-mentioned project, wherein aforementioned substituted or unsubstituted alkyl is selected from by taking Generation or unsubstituted linear or branched alkyl group, substituted or unsubstituted linear chain or branched chain alkenyl, substituted or unsubstituted straight chain or Branch alkynyl, substituted or unsubstituted naphthenic base, substituted or unsubstituted cycloalkenyl, substituted or unsubstituted aryl, substitution or The group of the group of unsubstituted linear chain or branched chain aryl alkyl composition.
(project 15)
The method according to any one of above-mentioned project, wherein aforementioned substituted or unsubstituted linear or branched alkyl group For linear chain or branched chain halogenated alkyl.
(project 16)
The method according to any one of above-mentioned project, wherein aforementioned linear or branched haloalkyl are straight chain or branch Chain perfluoroalkyl.
(project 17)
It is a kind of for manufacturing the kit of following general formula compound represented, wherein the general formula are as follows:
[chemical formula 13]
Wherein, in formula (13), each mark is identical as aforementioned meaning,
The kit includes:
[chemical formula 11]
[chemical formula 12]
With
P(OR3)(OR3’) OH,
In formula (11), R2Indicating the protecting group of hydrogen atom or hydroxyl, B indicates nucleic acid base optionally with protecting group,
In formula (12), R1It indicates selected from the group being made of substituted or unsubstituted aryl and substituted or unsubstituted heteroaryl Group, X is halogen atom,
Formula P (OR3)(OR3’) in OH, R3And R3’It separately indicates selected from by substituted or unsubstituted aryl and substitution Or the group of the group of unsubstituted heteroaryl composition.
(project 18)
According to kit described in above-mentioned project, wherein optionally the nucleic acid base with aforementioned protecting group is selected from by as follows The group of composition,
[chemical formula 14]
(in formula (14), R3a、R3bAnd R3cIt separately indicates selected from by hydrogen atom, substituted or unsubstituted straight chain or branch Alkanoyl, substituted or unsubstituted alkyl, substituted or unsubstituted naphthenic base, takes substituted or unsubstituted aryl-acyl The group or R of the group of generation or unsubstituted aryl and substituted or unsubstituted heteroaryl composition3aWith R3bIt is formed together amidine type The group of protecting group).
(project 19)
The kit according to any one of above-mentioned project, wherein aforementioned amidine type protecting group is N, N- dimethyl carbonamidine Base or N, N- dimethyl ethanamidine base.
(project 20)
The kit according to any one of above-mentioned project, wherein R1For substituted or unsubstituted aryl.
(project 21)
The kit according to any one of above-mentioned project, wherein aforementioned substituted or unsubstituted aryl is phenyl.
(project 22)
The kit according to any one of above-mentioned project, wherein the protecting group of aforementioned hydroxy is selected from being protected by ether system Protect the protecting group of the group of base, silyl ether system protecting group, acetal system protecting group and acyl group system protecting group composition.
(project 23)
The kit according to any one of above-mentioned project, wherein aforementioned ether system protecting group are as follows:
[chemical formula 15]
(in formula (R2a), R4a1、R4a2、R4a3、R4a4、R4a5、R4b1、R4b2、R4b3、R4b4、R4b5、R4c1、R4c2、R4c3、R4c4With R4c5Separately for selected from by hydrogen atom, substituted or unsubstituted straight or branched alkoxyl and substituted or unsubstituted straight The group of the group of chain or branched alkyl composition.).
(project 24)
The kit according to any one of above-mentioned project, wherein aforementioned substituted or unsubstituted linear chain or branched chain alkane Oxygroup is linear chain or branched chain perfluoro alkoxy.
(project 25)
The kit according to any one of above-mentioned project, wherein aforementioned substituted or unsubstituted linear chain or branched chain alkane Base is linear chain or branched chain perfluoroalkyl.
(project 26)
The kit according to any one of above-mentioned project, wherein aforementioned silyl ether system protecting group are as follows:
[chemical formula 16]
(in formula (R2b), R4d、R4eAnd R4fSeparately for selected from by substituted or unsubstituted linear or branched alkyl group, The group of the group of substituted or unsubstituted aryl and substituted or unsubstituted alkenyl composition.).
(project 27)
The kit according to any one of above-mentioned project, wherein aforementioned substituted or unsubstituted alkenyl is allyl.
(project 28)
The kit according to any one of above-mentioned project, wherein aforementioned silyl ether system protecting group are as follows:
[chemical formula 17]
(in formula (R2c), R4g、R4hAnd R4iSeparately for selected from not taking by substituted or unsubstituted alkyl, substitution or The group of the group of the silicyl in generation and substituted or unsubstituted silicyl oxygroup alkyl composition.).
(project 29)
The kit according to any one of above-mentioned project, wherein aforementioned silicyl is replaced by 1~3 alkyl.
(project 30)
The kit according to any one of above-mentioned project, wherein aforementioned substituted or unsubstituted alkyl be selected from by Substituted or unsubstituted linear or branched alkyl group, substituted or unsubstituted linear chain or branched chain alkenyl, substituted or unsubstituted straight chain Or branch alkynyl, substituted or unsubstituted naphthenic base, substituted or unsubstituted cycloalkenyl, substituted or unsubstituted aryl, substitution Or the group of the group of unsubstituted linear chain or branched chain aryl alkyl composition.
(project 31)
The kit according to any one of above-mentioned project, wherein aforementioned substituted or unsubstituted linear chain or branched chain alkane Base is linear chain or branched chain halogenated alkyl.
(project 32)
The kit according to any one of above-mentioned project, wherein aforementioned linear or branched haloalkyl be straight chain or Branched chain perfluoroalkyl group.
(project 33)
It is a kind of for selectively manufacturing the phosphorus reagent of following general formula compound represented or its salt or its solvate, In, the general formula are as follows:
[chemical formula 18]
(in formula (18), R1It indicates selected from being made of substituted or unsubstituted aryl and substituted or unsubstituted heteroaryl The group of group, R2Indicate the protecting group of hydrogen atom or hydroxyl, B indicates nucleic acid base optionally with protecting group)
The phosphorus reagent includes P (OR3)(OR3’) OH,
(formula P (OR3)(OR3’) in OH, R3And R3’It separately indicates selected from by substituted or unsubstituted aryl and substitution Or the group of the group of unsubstituted heteroaryl composition).
(project 34)
According to phosphorus reagent described in above-mentioned project, wherein optionally the nucleic acid base with aforementioned protecting group is selected from by as follows The group of composition,
[chemical formula 19]
(in formula (19), R3a、R3bAnd R3cIt separately indicates selected from by hydrogen atom, substituted or unsubstituted straight chain or branch Alkanoyl, substituted or unsubstituted alkyl, substituted or unsubstituted naphthenic base, takes substituted or unsubstituted aryl-acyl The group or R of the group of generation or unsubstituted aryl and substituted or unsubstituted heteroaryl composition3aWith R3bIt is formed together amidine type The group of protecting group).
(project 35)
The phosphorus reagent according to any one of above-mentioned project, wherein aforementioned amidine type protecting group is N, N- dimethyl carbonamidine Base or N, N- dimethyl ethanamidine base.
(project 36)
The phosphorus reagent according to any one of above-mentioned project, wherein R1For substituted or unsubstituted aryl.
(project 37)
The phosphorus reagent according to any one of above-mentioned project, wherein aforementioned substituted or unsubstituted aryl is phenyl.
(project 38)
The phosphorus reagent according to any one of above-mentioned project, wherein the protecting group of aforementioned hydroxy is selected from being protected by ether system Protect the protecting group of the group of base, silyl ether system protecting group, acetal system protecting group and acyl group system protecting group composition.
(project 39)
The phosphorus reagent according to any one of above-mentioned project, wherein aforementioned ether system protecting group are as follows:
[chemical formula 20]
(in formula (R2a), R4a1、R4a2、R4a3、R4a4、R4a5、R4b1、R4b2、R4b3、R4b4、R4b5、R4c1、R4c2、R4c3、R4c4With R4c5Separately for selected from by hydrogen atom, substituted or unsubstituted straight or branched alkoxyl and substituted or unsubstituted straight The group of the group of chain or branched alkyl composition.).
(project 40)
The phosphorus reagent according to any one of above-mentioned project, wherein aforementioned substituted or unsubstituted linear chain or branched chain alkane Oxygroup is linear chain or branched chain perfluoro alkoxy.
(project 41)
The phosphorus reagent according to any one of above-mentioned project, wherein aforementioned substituted or unsubstituted linear chain or branched chain alkane Base is linear chain or branched chain perfluoroalkyl.
(project 42)
The phosphorus reagent according to any one of above-mentioned project, wherein aforementioned silyl ether system protecting group are as follows:
[chemical formula 21]
(in formula (R2b), R4d、R4eAnd R4fSeparately for selected from by substituted or unsubstituted linear or branched alkyl group, The group of the group of substituted or unsubstituted aryl and substituted or unsubstituted alkenyl composition.).
(project 43)
The phosphorus reagent according to any one of above-mentioned project, wherein aforementioned substituted or unsubstituted alkenyl is allyl.
(project 44)
The phosphorus reagent according to any one of above-mentioned project, wherein aforementioned silyl ether system protecting group are as follows:
[chemical formula 22]
(in formula (R2c), R4g、R4hAnd R4iSeparately for selected from not taking by substituted or unsubstituted alkyl, substitution or The group of the group of the silicyl in generation and substituted or unsubstituted silicyl oxygroup alkyl composition.).
(project 45)
The phosphorus reagent according to any one of above-mentioned project, wherein aforementioned silicyl is replaced by 1~3 alkyl.
(project 46)
The phosphorus reagent according to any one of above-mentioned project, wherein aforementioned substituted or unsubstituted alkyl be selected from by Substituted or unsubstituted linear or branched alkyl group, substituted or unsubstituted linear chain or branched chain alkenyl, substituted or unsubstituted straight chain Or branch alkynyl, substituted or unsubstituted naphthenic base, substituted or unsubstituted cycloalkenyl, substituted or unsubstituted aryl, substitution Or the group of the group of unsubstituted linear chain or branched chain aryl alkyl composition.
(project 47)
The phosphorus reagent according to any one of above-mentioned project, wherein aforementioned substituted or unsubstituted linear chain or branched chain alkane Base is linear chain or branched chain halogenated alkyl.
(project 48)
The phosphorus reagent according to any one of above-mentioned project, wherein aforementioned linear or branched haloalkyl be straight chain or Branched chain perfluoroalkyl group.
(project 49)
A kind of P (OR3)(OR3’) OH is for selectively manufacturing following general formula compound represented or its salt or its solvent Application in compound, wherein the general formula are as follows:
[chemical formula 22A]
(in formula (22A), R1It indicates selected from being made of substituted or unsubstituted aryl and substituted or unsubstituted heteroaryl The group of group, R2Indicate the protecting group of hydrogen atom or hydroxyl, B indicates nucleic acid base optionally with protecting group),
(formula P (OR3)(OR3’) in OH, R3And R3’It separately indicates selected from by substituted or unsubstituted aryl and substitution Or the group of the group of unsubstituted heteroaryl composition).
In the present invention, for said one or multiple features, other than the combination having revealed that, it may also attempt to further Combination is to provide.Those skilled in the art read detailed description below then it is to be realized that other implementations of the invention as needed Mode and advantage.
The effect of invention
In accordance with the invention it is possible to be manufactured with low cost ribonucleic acid H- phosphate ester monomer, as a result, it is possible to it is low at This manufacture RNA.It is a feature of the present invention that passing through the hydroxyl and aromatic series carboxylic acid halides of the 2 ' positions and 3 ' positions that keep reactivity slightly different It reacts at low temperature, to carry out the esterification of 2 ' positions selectivity, then passes through the hydroxyl using 3 ' positions in one pot of phosphono capture Base, the rearrangement of 2 ' positions and 3 ' positions without causing acyl group.
Detailed description of the invention
Fig. 1 shows 5 '-HO-rU19The LC-MS of dT-3 '-OH analyzes result.
Fig. 2 shows 5 '-HO-rU3rCrGrArU3The LC-MS of dT-3 '-OH analyzes result.
Specific embodiment
To show best mode side below, the present invention will be described.Throughout the specification, the expression of singular exists When there is no special declaration, it is thus understood that also include the concept of its plural form.Therefore, the article of singular is (for example, English Situation " a ", " an ", " the " etc.) when there is no special declaration, it is thus understood that it also include the concept of its plural form.In addition, this Terminology used herein is not when having special declaration, it is thus understood that is used with the usually used in this field meaning. Therefore, as long as no other meanings are defined as, then whole technical term used in this specification and scientific and technical term have The identical meaning is generally understood with the those skilled in the art in field belonging to the present invention.In contradictory situation, this specification (including definition) is preferential.
(definition of term)
The term in this specification is illustrated below.
" substitution " refers in this specification: by some specific hydrogen atom other atoms or atomic group of organic compound Replace.The imported atom of hydrogen atom or atomic group will be replaced to be known as " substituent group ".Functional group can be considered as substituent group.As The example of substituent group, such as can enumerate: halogen atom, hydroxyl, alkyl, alkenyl, alkynyl, naphthenic base, aryl or heteroaryl etc..
" halogen atom " includes fluorine atom, chlorine atom, bromine atom and iodine atom.
" hydroxyl " refers in this specification: group shown in-OH.
Refer in this specification for the group of " straight chain ": constituting the carbon of the group both or does not have without branch and with forming ring Connect into the group of linear shape.
Refer in this specification for the group of " branch ": the carbon atom with 2 or more carbon atoms and the composition group The group of the structure of bonding.
In the present specification, C1, C2, Cn indicate that (herein, n indicates arbitrary positive integer to carbon number.).Therefore, C1 is used for table Show the substituent group of 1 carbon number.
" alkyl " refers in this specification: losing a hydrogen from the aliphatic hydrocarbon (alkane) of methane, ethane, propane etc Atom and the group of 1 valence generated, usually by CnH2n+1It indicates (herein, n is positive integer).Such as it can enumerate: methyl, second Base, n-propyl, isopropyl, normal-butyl, isobutyl group, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, dissident Base, n-heptyl, different heptyl, n-octyl, iso-octyl, n-nonyl, positive decyl etc.." replace alkyl " in this specification to refer to: alkyl The alkyl that is replaced by above-mentioned defined substituent group of H.
" alkenyl " refers in this specification: losing one from the aliphatic hydrocarbon (alkene) in the molecule with a double bond Hydrogen atom and the group of 1 valence generated, usually by CnH2n-1Indicate (herein, the positive integer that n is 2 or more).Such as it can enumerate: Vinyl, allyl, acrylic, isopropenyl, cyclobutenyl, isobutenyl, isopentene group (Prenyl), butadienyl, amylene Base, isopentene group (Isopentenyl), pentadienyl, hexenyl, isohexenyl, hexadienyl, heptenyl, octenyl, nonene Base, decene base, undecenyl, dodecenyl succinic, tridecylene base, tetradecene base, pentadecane alkenyl etc.." substituted alkenyl " Refer to: the alkenyl that the H of alkenyl is replaced by above-mentioned defined substituent group.
" allyl " refers in this specification: CH2=CH-CH2Shown in group.
" alkynyl " refers in this specification: losing one from the aliphatic hydrocarbon (alkynes) in the molecule with three keys Hydrogen atom and the group of 1 valence generated, usually by CnH2n-3Indicate (herein, the positive integer that n is 2 or more).For example, acetylene Base, propinyl, butynyl, pentynyl, hexin base, heptynyl, octynyl, n-heptylacetylene base, decynyl etc.." substituted alkynyl " refers to: The alkynyl that the H of alkynyl is replaced by above-mentioned defined substituent group.
" naphthenic base " refers in this specification: the alkyl with ring structures.Such as can enumerate: cyclopropyl, cyclobutyl, Cyclopenta, cyclohexyl, suberyl, cyclooctyl, cyclononyl, cyclodecyl etc.." substituted cycloalkyl " refers to: the H of naphthenic base is above-mentioned The naphthenic base that defined substituent group replaces.
" cycloalkenyl " refers in this specification: the alkenyl with ring structures.Such as it can enumerate: cyclopropanyl, ring fourth Alkenyl, cyclopentenyl, cyclohexenyl group, cycloheptenyl, cyclo-octene base, cyclonoene base, cyclodecene base etc.." substituted cycloalkenyl " is Refer to: the cycloalkenyl that the H of cycloalkenyl is replaced by above-mentioned defined substituent group.
" aryl " refers in this specification: making the group of 1 hydrogen atom being bonded with the ring of aromatic hydrocarbon disengaging and generation. By benzenesulfonamide derivative phenyl (C6H5), by the derivative tolyl (CH of toluene3C6H4), by diformazan benzenesulfonamide derivative xylyl ((CH3)2C6H3), by the derivative naphthalene (C of naphthalene10H8), by the derivative phenanthryl (C of phenanthrene14H9), by the derivative anthryl (C of anthracene14H9), spread out by aphthacene Raw aphthacene base (C18H11), base (C is bent by Qu Yansheng18H11), by the derivative pyrenyl (C of pyrene18H11), by the derivative benzo of BaP Pyrenyl (C20H11), by the derivative pentacene (C of pentacene22H13-)。
" heteroaryl " refers in this specification: in ring with 1 any of the above identical in O, S and N or not Aryl more than together heteroatomic, monocycle or two rings.Heteroaryls more than two rings further includes miscellaneous more than monocycle or two rings Group made of the ring in above-mentioned " aryl " has been condensed on aryl.
As the heteroaryl of monocycle, preferably 5~8 yuan, more preferably 5- or 6-membered.Such as it can enumerate: pyrrole radicals, imidazoles Base, pyrazolyl, pyridyl group, pyridazinyl, pyrimidine radicals, pyrazinyl, triazolyl, triazine radical, tetrazole radical, furyl, thienyl, different evil Oxazolyl, oxazolyl, oxadiazoles base, isothiazolyl, thiazolyl, thiadiazolyl group etc..
It as the heteroaryl of two rings, such as can enumerate: indyl, isoindolyl, indazolyl, indolizine base, quinolyl, different Quinolyl, cinnoline base, phthalazinyl, quinazolyl, phthalazinyl, quinoxalinyl, purine radicals, pteridyl, benzimidazolyl, benzene And isoxazolyl, benzoxazolyl, benzoxadiazole base, benzisothia oxazolyl, benzothiazolyl, diazosulfide base, benzo Furyl, isobenzofuran-base, benzothienyl, benzotriazole base, imidazopyridyl, triazolo pyridyl, imidazo thiophene Oxazolyl, pyrazine and pyridazinyl, azoles and pyridyl group, thiazolopyridinyl etc..
It as heteroaryl more than tricyclic, such as can enumerate: carbazyl, acridinyl, xanthyl, phenothiazinyl, phenoxazine Thiophene base, phenazinyl, dibenzofuran group etc..
" alkoxy " refers in this specification: the group of above-mentioned " alkyl " and oxygen atoms bond.Such as it can enumerate: methoxy Base, ethyoxyl, positive propoxy, isopropoxy, n-butoxy, tert-butoxy, isobutoxy, sec-butoxy, amoxy, isoamyl oxygen Base, hexyl oxygroup etc..
" halogenated alkyl " refers in this specification: the base that 1 or more above-mentioned " halogen atom " is bonded with above-mentioned " alkyl " Group.Such as it can enumerate: single methyl fluoride, single fluoro ethyl, single fluoropropyl, 2,2,3,3,3- pentafluoropropyl group, monochloro methyl, fluoroform Base, trichloromethyl, 2,2,2- trifluoroethyl, 2,2,2- trichloroethyl, 1,2- dibromoethyl, 1,1,1- trifluoro propane -2- base etc..
" perfluoroalkyl " refers in this specification: all or part of hydrogen with the bond with carbon of alkyl is by fluorine-substituted alkane Base.
" perfluoro alkoxy " refers in this specification: fluorine-substituted with all or part of hydrogen of the bond with carbon of alkoxy Alkoxy.
" alkyl acyl " refers in this specification: the group of above-mentioned " alkyl " and carbonyl linkage.Such as it can enumerate: methyl Carbonyl, ethylcarbonyl group, propyl carbonyl, Isopropylcarbonyl, tert-butyl carbonyl, butylcarbonyl, sec-butylcarbonyl group, pentylcarbonyl, Isopentyl carbonyl, hexyl carbonyl etc..
" aryl-acyl " refers in this specification: the group of above-mentioned " aryl " and carbonyl linkage.Such as it can enumerate: phenyl Carbonyl, naphthalene carbonyl, luxuriant and rich with fragrance Epoxide carbonyl, anthrylcarbonyl etc..
" aryl alkyl " refers in this specification: the alkyl replaced by 1 or more above-mentioned " aryl ".Such as it can enumerate Out: benzyl, phenethyl, phenylpropynyl, benzhydryl, trityl, menaphthyl etc..
" silicyl " refers in this specification: 3 and silicon in above-mentioned " alkyl ", " alkenyl ", " alkynyl " or " aryl " The group of atomistic binding.It herein can be identical or different with the group of 3 silicon atom bondings.Such as it can enumerate: trimethyl first Silylation (TMS), triethylsilyl (TES), t-butyldimethylsilyl (TBDMS), triisopropylsilyl (TIPS), t-butyldiphenylsilyl (TBDPS) etc..
" silicyl oxygroup alkyl " refers in this specification: the hydrogen on above-mentioned " alkyl " is by as above-mentioned " silicyl " The group replaced with " the silicyl oxygroup " of the group of oxygen atoms bond.Such as it can enumerate: trimethyl silyl oxygroup Methyl, triethylsilyl oxygroup ethyl, t-butyldimethylsilyl oxygroup methyl, triisopropylsilyl oxygroup Propyl, t-butyldiphenylsilyl oxygroup butyl etc., but not limited thereto.
" cation " refers in this specification: the ion with positive charge such as can enumerate: hydrogen ion, quaternary ammonium ion, Lithium ion, sodium ion, potassium ion etc., but not limited thereto.
" tertiary amine " refers in this specification: the compound that whole hydrogen of ammonia are replaced by above-mentioned " alkyl " or above-mentioned " aryl ". Such as can enumerate: triethylamine, n,N-diisopropylethylamine, tripropyl amine (TPA) etc., but not limited thereto.
" nucleic acid base " refers in this specification: constituting the base content of nucleic acid, such as can enumerate: adenine (A), bird Purine (G), cytimidine (C), thymidine (T), uracil (U) etc., but not limited thereto.
" oligomer " refers in this specification: the degree of polymerization of the polymer of low polymerization degree, oligomer is not limited to this, is 2 ~100.
" protecting group " refers in this specification: for protecting functional group from specifically chemically reacting the base for influencing and using Group.
As the protecting group of hydroxyl, can enumerate: ether system protecting group (methyl (Me), benzyl (Bn), to methoxy-benzyl (PMB), tert-butyl (t-Bu), trityl (Tr) etc.), silyl ether system protecting group (trimethyl silyl (TMS), three Ethyl silicane base (TES), t-butyldimethylsilyl (TBDMS), triisopropylsilyl (TIPS), tert-butyl two Phenyl silyl groups (TBDPS) etc.), acetal system protecting group (methoxy (MOM), 2- tetrahydrofuran base (THP), ethyoxyl Ethyl (EE) etc.), acyl group system protecting group (acetyl group (Ac), pivaloyl group (Piv), benzoyl (Bz) etc.) etc., but do not limit In this.
It as the protecting group of amino, can enumerate: carbamate system protecting group (tert-butoxycarbonyl (Boc), benzyl oxygen Base carbonyl (Cbz), 9- fluorenylmethyloxycarbonyl (Fmoc), 2,2,2- tri-chloroethoxy base carbonyl (Troc), allyl oxygroup carbonyl Base (Alloc) etc.), amide system protecting group (trifluoroacetyl group etc.), imide series protecting group (phthalyl (Pht) etc.), Sulphonyl amine system protecting group (p-toluenesulfonyl (Ts), 2- nitrobenzenesulfonyl (Ns) etc.), amidine type protecting group (N, N- dimethyl methyl Amidino groups, N, N- dimethyl ethanamidine base etc.) etc., but not limited thereto.
" amidine type protecting group " refers in this specification: protecting group shown in general formula below,
[chemical formula 23]
(in formula (23), R1、R2And R3Separately for selected from not taking by hydrogen, substituted or unsubstituted alkyl, substitution or The group of the naphthenic base in generation, the group of substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl composition)
For example, can enumerate: N, N- dimethylformamidinyl, N, N- dimethyl ethanamidine base etc., but not limited thereto.
In the present specification, " solvation " refers to: some solvent molecules are surrounded by solute molecule or ion and become one The phenomenon that group.
The compound of the present invention can also provide in a salt form.Such as the salt of organic base (triethylamine etc.) can be enumerated. The salt with triethylamine can especially be enumerated.It can use the method usually carried out and form these salt.Or manufacture according to the present invention The condition of method can generate compound (being e.g. originated from the substance of additive) in a salt form.
The compound of the present invention can also be provided in the form of solvate.Such as can enumerate with water, tertiary amine solvent Compound.Such solvate can use the method that usually carries out to be formed.Or the condition of manufacturing method according to the invention, Compound can be generated in the form of solvate.In turn, the salt of above compound can be generated simultaneously in the form of solvate It provides.
In the present specification, " reaction vessel " refers to: the device chemically reacted in the manufacturing process of chemical substance, Such as can enumerate: flask, beaker etc., but not limited thereto.
In the present specification, " Component units " refer to: constituting the atom or atom of a part of the basic structure of oligomer Group, synonymously uses with " monomer ".
(explanation of preferred embodiment)
The preferred embodiment of the present invention will be described below.Embodiment provided below is for preferably The example for understanding the present invention and providing, it will be appreciated that should not be limited to record below for the scope of the present invention.Therefore, this field skill It is obvious that it is suitable for change that art personnel, which can refer to the record in this specification and carry out within the scope of the invention,.Separately Outside, it will be appreciated that for the following embodiments and the accompanying drawings of the invention can be used alone or combine they come using.
In a mode, the present invention provides a kind of manufacture general formula compound represented below or its salt or its solvation The method of object, the general formula are as follows:
[chemical formula 24]
(in formula (24), R1It indicates selected from being made of substituted or unsubstituted aryl and substituted or unsubstituted heteroaryl The group of group, R2Indicate the protecting group of hydrogen atom or hydroxyl, B indicates nucleic acid base optionally with protecting group)
This method includes process 1 and process 2,
Process 1 is to make [chemical formula 25]
It is reacted with [chemical formula 26],
(in formula (26), X is halogen atom)
And the process for obtaining [chemical formula 27],
Process 2 is to make [chemical formula 28]
With P (OR3)(OR3’) OH reaction,
And the process for obtaining [chemical formula 29],
(formula P (OR3)(OR3’) in OH, R3And R3’It separately indicates selected from by substituted or unsubstituted aryl and substitution Or the group of the group of unsubstituted heteroaryl composition),
The process 1 and the process 2 carry out in 1 reaction vessel.
In the past, when selectively obtaining 2 ' positions protection ribonucleotide monomer by organic synthesis, it is known that as follows Shown in formula like that,
[chemical formula 30]
After the hydroxyl of 2 ' positions imports protecting group, the hydroxyl using phosphorus reagent in 3 ' positions imports method as phosphate. When carrying out above-mentioned synthesis, the protected intermediate of hydroxyl (B) of the protected intermediate of the hydroxyl of 2 ' positions (A) and 3 ' positions is in flat Weighing apparatus relationship, the molar ratio of intermediate (A) and intermediate (B) reach 50/50 as time goes by, it is therefore desirable to promptly be led Enter the reaction of phosphate.Therefore it needs using the very high phosphorus reagent of reactivity.
However, very high the being usually toxic property of phosphorus reagent of reactivity is high, it is unstable at room temperature to wait operating difficulties.In addition, There is also being difficult to remove reagent residue (such as phosphoric acid) according to phosphorus reagent, need to increase number of column chromatography etc. in purifying Time-consuming such problems point.
By using the relatively low phosphate of reactivity as phosphorus reagent in the present invention, thus it was unexpectedly observed that 2 ' The hydroxyl of position is selectively introduced protecting group and can easily remove the residue of phosphate from reaction product.And then due to that can be easy The residue of phosphate is removed, therefore compared with the conventional method improves also the purity of 2 ' positions protection ribonucleotide monomer.
In addition, since the substrate specificity than using the method for existing enzyme is wide, it is thus possible to synthesize a greater variety of 2 ' Position protection ribonucleotide monomer.
In one preferred embodiment, process 1 carries out in a low temperature of being lower than room temperature, and reaction temperature at this time is- 40~20 DEG C, -40~15 DEG C, -40~10 DEG C, -40~5 DEG C, -40~0 DEG C, -40~-10 DEG C, -40~-20 DEG C, -40~-30 DEG C, -30~20 DEG C, -20~20 DEG C, -10~20 DEG C, 0~20 DEG C, 5~20 DEG C, however, not limited to this, preferably -40~20 DEG C Under.Although undesirable be bound by theory, reason is that, according to base, solvent, intermediate (A) is different with intermediate (B) Structureization reaction carries out, thus intermediate (A) and the molar ratio of intermediate (B) is made to reach 50/50.It should be noted that even if In the case where more than room temperature being reacted, isomerization can't also occur within the shorter time (a few hours), it was confirmed that in spy Above-mentioned isomerization reaction when room temperature or more is maintained under fixed condition (such as when concentration) to carry out.In addition, even if isomerization reaction It carries out, if 10% or so, then can be removed after phosphorylation by purifying, therefore can receive.
In one preferred embodiment, after process 1 within 1 minute, within 2 minutes, within 3 minutes, 4 points Within clock, within 5 minutes, within 10 minutes, within 20 minutes, within 30 minutes, within 40 minutes, within 50 minutes, 1 hour Within, within 2 hours, within 3 hours, within 4 hours carry out process 2, preferably carry out work within 1 hour after process 1 Sequence 2.Whether process 1 terminates to can use thin-layered chromatography (TLC), liquid chromatography to be confirmed, but not limited thereto.Separately Outside, in advance carry out preliminary experiment estimation arrive process 1 terminate time in the case where, can not to process 1 whether terminate into Process 2 is carried out under the premise of row confirmation.
In one preferred embodiment, carry out process 2 when reaction system in temperature be -40~25 DEG C, -40~ 20 DEG C, -40~10 DEG C, -40~5 DEG C, -40~0 DEG C, -40~-10 DEG C, -40~-20 DEG C, -40~-30 DEG C, -30~25 DEG C, - 20~25 DEG C, -10~25 DEG C, 0~25 DEG C, 5~25 DEG C, 10~25 DEG C, 20~25 DEG C, -30~20 DEG C, -20~20 DEG C, -10 ~20 DEG C, 0~20 DEG C, 5~20 DEG C, 10~20 DEG C, however, not limited to this, preferably -20~20 DEG C.
In one preferred embodiment, alkali preferably is added before adding phosphite ester in process 2.While it is desirable to not Bound by theory, but this is for quenching the added excessive carboxylic acid halides in process 1.The preferred dimethylamine salt of the alkali added at this time Hydrochlorate, but not limited thereto.Until the time of addition phosphite ester is 30 minutes, 1 hour, 2 hours, 3 hours, 4 after addition alkali Hour, but not limited thereto.In addition, addition alkali when temperature be -40 DEG C~room temperature, -40~30 DEG C, -40~20 DEG C, -40~ 10 DEG C, -40~5 DEG C, -40~0 DEG C, -40~-10 DEG C, -40~-20 DEG C, -40~-30 DEG C, -30~30 DEG C, -20~30 DEG C, - 10~30 DEG C, 0~30 DEG C, 5~30 DEG C, 10~30 DEG C, 20~30 DEG C, however, not limited to this, preferably -40 DEG C~room temperature, -40 ~0 DEG C.
Product obtained from reaction through the invention is for purity before purification also than passing through existing reaction Obtained product is high, therefore purifying later easier than the product as obtained from existing reaction can also carry out.
In one preferred embodiment, optionally the nucleic acid base with aforementioned protecting group is selected from by forming as follows Group,
[chemical formula 31]
(in formula (31), R3a、R3bAnd R3cIt separately indicates selected from by hydrogen atom, substituted or unsubstituted straight chain or branch Alkanoyl, substituted or unsubstituted alkyl, substituted or unsubstituted naphthenic base, takes substituted or unsubstituted aryl-acyl The group or R of the group of generation or unsubstituted aryl and substituted or unsubstituted heteroaryl composition3aWith R3bIt is formed together amidine type The group of protecting group), in further preferred embodiment, aforementioned amidine type protecting group is N, N- dimethylformamidinyl or N, N- Dimethyl ethanamidine base.In reaction of the invention, not only when nucleic acid base is purine bases, selected when for pyrimidine bases Carried out in 2 ' positions to selecting property.
In one preferred embodiment, R1For substituted or unsubstituted aryl, in further preferred embodiment In, aforementioned substituted or unsubstituted aryl is phenyl.Although undesirable be bound by theory, reason is carrying out process It is selectively reacted when 1 reaction in 2 ' positions.
In one preferred embodiment, the protecting group of aforementioned hydroxy is selected from by ether system protecting group, silyl ether It is the protecting group of the group of protecting group, acetal system protecting group and acyl group system protecting group composition.Even if using arbitrary 2 ' position of protecting group Also it is selectively reacted.
In further preferred embodiment, aforementioned ether system protecting group are as follows:
[chemical formula 32]
(in formula (R2a), R4a1、R4a2、R4a3、R4a4、R4a5、R4b1、R4b2、R4b3、R4b4、R4b5、R4c1、R4c2、R4c3、R4c4With R4c5Separately for selected from by hydrogen atom, substituted or unsubstituted straight or branched alkoxyl and substituted or unsubstituted straight The group of the group of chain or branched alkyl composition.).Although undesirable be bound by theory.But by changing the substitution on phenyl Base and disengaging ability can be adjusted.
In further preferred embodiment, aforementioned substituted or unsubstituted straight or branched alkoxyl is straight chain or branch Chain perfluoro alkoxy, in addition, aforementioned substituted or unsubstituted linear or branched alkyl group is linear chain or branched chain perfluoroalkyl.Although no It is expected that being bound by theory, but the reason is that, easy separation/purifying remove-insurance is able to carry out by using the solvent containing fluorine Protecting group and target product after shield.
In further preferred embodiment, aforementioned silyl ether system protecting group are as follows:
[chemical formula 33]
(in formula (R2b), R4d、R4eAnd R4fSeparately for selected from by substituted or unsubstituted linear or branched alkyl group, The group of the group of substituted or unsubstituted aryl and substituted or unsubstituted alkenyl composition.).In further preferred embodiment In, aforementioned substituted or unsubstituted alkenyl is allyl.Although undesirable be bound by theory, by changing on silicon atom Substituent group and disengaging ability can be adjusted.
In further preferred embodiment, aforementioned silyl ether system protecting group are as follows:
[chemical formula 34]
(in formula (R2c), R4g、R4hAnd R4iSeparately for selected from not taking by substituted or unsubstituted alkyl, substitution or The group of the group of the silicyl in generation and substituted or unsubstituted silicyl oxygroup alkyl composition.), in further preferred reality It applies in mode, aforementioned silicyl is replaced by 1~3 alkyl.It is undesirable to be bound by theory, but by changing substituent group Disengaging ability can be adjusted.
In further preferred embodiment, aforementioned substituted or unsubstituted alkyl is selected from by substituted or unsubstituted Linear or branched alkyl group, substituted or unsubstituted linear chain or branched chain alkynyl, replaces substituted or unsubstituted linear chain or branched chain alkenyl Or unsubstituted naphthenic base, substituted or unsubstituted cycloalkenyl, substituted or unsubstituted aryl, substituted or unsubstituted straight chain or The group of the group of branch aryl alkyl composition.
In further preferred embodiment, aforementioned substituted or unsubstituted linear or branched alkyl group is linear chain or branched chain Halogenated alkyl, in further preferred embodiment, aforementioned linear or branched haloalkyl are linear chain or branched chain perfluoroalkyl. Although undesirable be bound by theory, reason is easily be separated/be purified by using the solvent containing fluorine de- Protecting group and target product after protection.
In a mode, the present invention is provided to manufacture the kit of following general formula compound represented, wherein described General formula are as follows:
[chemical formula 37]
(in formula (37), each mark is identical as aforementioned meaning)
The kit includes:
[chemical formula 35]
[chemical formula 36]
With
P(OR3)(OR3’) OH,
(in formula (35), R2Indicate the protecting group of hydrogen atom or hydroxyl, B indicates nucleic acid base optionally with protecting group),
(in formula (36), R1It indicates selected from being made of substituted or unsubstituted aryl and substituted or unsubstituted heteroaryl The group of group, X is halogen atom),
(formula P (OR3)(OR3’) in OH, R3And R3’It separately indicates selected from by substituted or unsubstituted aryl and substitution Or the group of the group of unsubstituted heteroaryl composition),
Although undesirable be bound by theory, reason is to state kit in use to synthesize 2 ' positions protection ribose core The case where glycosides monomer, due to that can be readily removable the residue of phosphorus reagent in purifying, and can be readily derived the 2 ' of purity is high Position protection ribonucleotide monomer.
In a mode, the present invention is provided to selectively manufacture following general formula compound represented or its salt or its The phosphorus reagent of solvate, wherein the general formula are as follows:
[chemical formula 38]
(in formula (38), R1It indicates selected from being made of substituted or unsubstituted aryl and substituted or unsubstituted heteroaryl The group of group, R2Indicate the protecting group of hydrogen atom or hydroxyl, B indicates nucleic acid base optionally with protecting group),
The phosphorus reagent includes P (OR3)(OR3’) OH,
(formula P (OR3)(OR3’) in OH, R3And R3’It separately indicates selected from by substituted or unsubstituted aryl and substitution Or the group of the group of unsubstituted heteroaryl composition).
Although undesirable be bound by theory, above-mentioned " selectively manufacturing " refers to: obtaining X product estimated In the case of, the ratio of the target product in reaction product entirety is 1/X or more or is greater than 1/X.Such as expect to obtain 2 products Situation refers to: the ratio of the target product in reaction product entirety be 50% (=1/2) more than or be greater than 50%, preferably 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or more Or it is bigger.
The general synthetic method of ribonucleic acid H- phosphate ester monomer
[chemical formula 39]
(in above-mentioned flow chart, PG1And PG2Indicate the protecting group of arbitrary hydroxyl, R3And R3’It indicates selected from by replacing or not The group of the group of substituted aryl and substituted or unsubstituted heteroaryl composition, B indicate nucleic acid base optionally with protecting group)
The synthesis of (1) 5 ' position protection nucleosides
Under non-active gas (such as nitrogen) atmosphere, in suitable reaction vessel (such as three-necked flask (500mL)) Nucleosides (such as adenosine 5.34g (20mmol)) and suitable reaction dissolvent (such as pyridine 400mL) is added, in suitable reaction temperature It is stirred under degree (such as 0 DEG C).The suitable protection reagent of addition (such as tertiary butyl chloride dimethylsilane 3.07g The dichloromethane solution 40mL of (20.4mmol)) (such as by being slowly added dropwise), under non-active gas (such as nitrogen) atmosphere, (such as 12 hours at room temperature) are stirred the suitable time at a suitable temperature.Distillation removal reaction dissolvent, it is residual what is obtained Water (such as 100mL) and organic solvent (such as chloroform 100mL/ methanol 40mL) are added in slag and is extracted.Vacuum distillation is gone Except (such as further with chloroform 100mL again after aqueous layer extracted) organic layer.Obtained mixture is carried out (such as using Silicagel column carries out) purifying, target fraction is concentrated under reduced pressure, target product (such as white solid) is thus obtained.
(2) synthesis of ribonucleic acid H- phosphate ester monomer
Under non-active gas (such as nitrogen) atmosphere, suitable reaction vessel (such as in three-necked flask (300mL) plus Enter 5 ' positions protection nucleosides (such as t-butyldimethylsilyl protection adenosine 3.82g (10mmol)) and suitable solvent (example Such as pyridine 100mL), it is stirred under suitable reaction temperature (such as -40 DEG C).The suitable protection reagent of addition (such as benzene The dichloromethane solution 10mL of formyl chloride 2.11g (15mmol)) (such as by being slowly added dropwise with 30 minutes), in suitable temperature The lower stirring suitable time (such as 3 hours at -40 DEG C).Suitable reagent (such as dimethylamine hydrochloric acid is added in reaction solution Salt 489mg (6mmol)), (such as disassembly cooling bath and from -40 DEG C to room temperature) temperature is improved, adds to stir after reagent and is suitble to Time (such as 2 hours).Suitable phosphorus reagent (such as diphenylphosphite is added at suitable temperature (such as room temperature) The pyridine solution 20mL of 7.03g (30mmol)) (such as by being slowly added dropwise), the suitable time is stirred at a suitable temperature (such as 3 hours at room temperature).After suitable alkali (such as triethylamine 10mL and water 10mL) is added, and then at a suitable temperature The stirring suitable time (such as 1 hour at room temperature).Vacuum distillation removal reaction dissolvent (such as pyridine, methylene chloride), Water (such as 100mL) is added in obtained residue, is carried out with organic solvent (such as chloroform 100mL) (such as twice) extract.Into The cleaning (such as triethylamine 4mL and water 100mL is added) for the organic layer that row obtains, then vacuum distillation removes organic solvent (example Such as chloroform).Carry out (such as in the crude product be added 0.5mL triethylamine, carried out using silicagel column) purifying, to target fraction into Row is concentrated under reduced pressure, and thus obtains target product (such as white solid).
(3) synthesis of oligonucleotides
Synthesis for oligonucleotides, in suitable reaction dissolvent (such as pyridine: MeCN=1:1 (0.05M)), suitable Starting base (5 '-OH-dT-3 '-O-suc-O-PEG), monomer (such as TBDMS- are used under the reaction temperature (such as 25 DEG C) of conjunction RU-H- phosphate (1.3 equivalent)), condensing agent (such as pivaloyl chloride (6.5 equivalent)), oxidant (such as 0.1M I2(1.5 work as Amount)), desiliconization alkylating agent (tetra-n-butyl ammonium fluoride (TBAF) (15 equivalent)) Lai Jinhang (such as passing through liquid phase synthesis) extend it is anti- It answers, in each extension, (such as being precipitated by ether) is purified.Pass through after extending to the desired degree of polymerization (such as degree of polymerization 20) Suitable reagent (such as ethylenediamine) carries out the deprotection of the protecting group of 2 ' position hydroxyls and the polymer of 3 ' position hydroxyls, then obtains (such as by carrying out further HPLC purifying) desired oligonucleotides.In turn, it is carried out by suitable reagent (such as TBAF) The deprotection of the protecting group (such as TBDMS yl) of 5 ' position hydroxyls, then obtains (such as purifying by the further progress HPLC) phase The oligonucleotides of prestige.
To the bibliography of the scientific literature, patent, patent application quoted in this manual etc., entire contents and each To same extent it is used as reference into this specification from the content specifically recorded.
Preferred embodiment has shown and described above for the present invention is readily appreciated that.Below based on embodiment to this Invention is illustrated, but above-mentioned explanation and embodiment below are given for example only and provide, be not intended to limit the present invention and It provides.Therefore, the scope of the present invention is both not limited to the embodiment specifically recorded in this specification or is not limited to implement Example, is only limited by claims.
Embodiment
The present invention is further illustrated according to embodiment comparative example below, but the not limited solution of the present invention It releases embodiment obtained from the technological means disclosed in these examples, each embodiment of proper combination and is also contained in model of the invention In enclosing.It should be noted that chemical combination name shown in embodiment below and comparative example is not necessarily named according to IUPAC Method.
In addition, abbreviation used in this specification indicates the meaning below.
Bu: butyl
Et: ethyl
D: deoxidation (DNA)
HPLC: high performance liquid chromatography
Me: methyl
NMR: nuclear magnetic resonance
PEG: polyethylene glycol
Ph: phenyl
R: ribose (RNA)
Suc: succinic acid
TBAF: tetra-n-butyl ammonium fluoride
TBDMS: t-butyldimethylsilyl
T-Bu: tert-butyl
THF: tetrahydrofuran
X: halogen group
NMR spectra is determined using Bruker corporation AVANCE III 400.
The condition research for the esterification that (reference example 1) has used 2 ' positions of adenosine to select
[chemical formula 40]
To the benzoylation of t-butyldimethylsilyl protection adenosine acetal tin (adenosine tin acetal) Condition research is carried out.The separation of 2 ' -ester and 3 ' -ester can be carried out by silicagel column, purify and asked using separation yield Generation ratio is gone out.
[table 1]
Table 1
When using benzoyl oxide as reagent, the protected ester body in 3 ' positions is preferentially selected.The case where having used chlorobenzoyl chloride, The protected ester body in preferential 2 ' position of selection target.If further selectively being reacted, using acetal tin, borate as promotion The case where reaction of agent, observes selective reduction.
Known to: isomerization is had occurred with molar ratio computing when room temperature is reaction solution to be concentrated under reduced pressure up and down, becomes 50/50.
According to the above results, by carrying out the synthesis of the phosphate body based on one kettle way, so as to solve isomerization Problem can obtain the adenosine monomer as target compound using good yield.
The synthesis of (embodiment 1) ribonucleic acid H- phosphate ester monomer
(1) synthetic method of adenosine monomer
[chemical formula 42]
The synthesis of (1-1) 5 '-t-butyldimethylsilyl adenosine
In a nitrogen atmosphere, adenosine 5.34g (20mmol) and pyridine 400mL are added in three-necked flask (500mL), 0 It is stirred at DEG C.The dichloromethane solution 40mL of tertiary butyl chloride dimethylsilane 3.07g (20.4mmol) is slowly added dropwise, in nitrogen To be stirred at room temperature 12 hours under gas atmosphere.Distillation removal pyridine and methylene chloride, are added water 100mL and chlorine in obtained residue Imitative 100mL/ methanol 40mL is simultaneously extracted.Further with after chloroform 100mL again aqueous layer extracted, vacuum distillation removal has Machine layer.Obtained mixture is purified using silicagel column, target fraction is concentrated under reduced pressure, 4.80g is thus obtained and (receives Rate 63%) white solid.
(1-2) 5 '-t-butyldimethylsilyl -2 '-- H- phosphate synthesis of benzoyl adenosine -3 '
In a nitrogen atmosphere, t-butyldimethylsilyl is added in three-necked flask (300mL) and protects adenosine 3.82g (10mmol) and pyridine 100mL, is stirred at -40 DEG C.The two of chlorobenzoyl chloride 2.11g (15mmol) is slowly added dropwise with 30 points Chloromethanes solution 10mL is stirred 3 hours at -40 DEG C in a nitrogen atmosphere.Dimethylamine hydrochloride 489mg is added in reaction solution (6mmol) dismantles cooling bath and temperature is increased to room temperature by -40 DEG C, stir 2 hours after adding dimethylamine hydrochloride.20 The pyridine solution 20mL of diphenylphosphite 7.03g (30mmol) is slowly added dropwise at DEG C, in a nitrogen atmosphere to be stirred at room temperature 3 Hour.After triethylamine 10mL and water 10mL is added, and then it is stirred at room temperature 1 hour.Vacuum distillation removal pyridine, dichloromethane Water 100mL is added in obtained residue, is extracted twice with chloroform 100mL for alkane.Triethylamine is added in obtained organic layer 4mL and water 100mL and after being cleaned, vacuum distillation eliminates chloroform.The triethylamine of 0.5mL is added in the crude product, utilizes Silicagel column carries out the purifying of target product, and target fraction is concentrated under reduced pressure, and it is solid thus to obtain 4.23g (yield 65%) white Body.
1H NMR(400MHz,CDCl3), σ 0.098 (6H, d), σ 0.907 (9H, s), σ 1.363 (9H, t, J=7.6Hz), σ (2.628-2.754 2H, m), σ 3.095 (6H, q, J=7.6Hz), σ 3.915 (2H, ddd), σ 4.341 (1H, q), σ 4.964- 5.007(1H,m),σ5.798(2H,br),σ6.546(1H,dd),σ6.975(1H,d,JPH=617Hz), σ 8.224 (1H, s), σ 8.324(1H,s).
31P NMR(162MHz,CDCl3),σ3.57
(2) synthetic method of uridine monomer
[chemical formula 43]
The synthesis of (2-1) 5 '-t-butyldimethylsilyl uridine
In a nitrogen atmosphere, uridine 4.88g (20mmol) and pyridine 40mL are added in three-necked flask (100mL), at 0 DEG C Under be stirred.The dichloromethane solution 40mL of tertiary butyl chloride dimethylsilane 3.07g (20.4mmol) is slowly added dropwise, in nitrogen To be stirred at room temperature 12 hours under atmosphere.Vacuum distillation removal pyridine and methylene chloride, are added water 100mL in obtained residue, It is extracted twice with chloroform 100mL.Vacuum distillation removal chloroform, is purified using silicagel column, depressurize to target fraction dense Contracting, thus obtains 5.59g (yield 78%) white solid.
(2-2) 5 '-t-butyldimethylsilyl -2 '-- H- phosphate synthesis of benzoyl uridine -3 '
In a nitrogen atmosphere, t-butyldimethylsilyl is added in three-necked flask (300mL) and protects uridine 3.58g (10mmol) and pyridine 100mL, is stirred at -40 DEG C.It was slowly added dropwise chlorobenzoyl chloride 2.11g's (15mmol) with 30 minutes Dichloromethane solution 10mL is stirred 3 hours with -40 DEG C in a nitrogen atmosphere.Dimethylamine hydrochloride 489mg is added in reaction solution (6mmol) dismantles cooling bath and temperature is increased to room temperature by -40 DEG C, stir 2 hours after adding dimethylamine hydrochloride.20 The pyridine solution 20mL of diphenylphosphite 7.03g (30mmol) is slowly added dropwise at DEG C, in a nitrogen atmosphere to be stirred at room temperature 3 Hour.Triethylamine 10mL and water 10mL is added, is stirred at room temperature 1 hour.Vacuum distillation removal pyridine, methylene chloride, To residue in be added water 100mL, be extracted twice with chloroform 100mL.Triethylamine 4mL and water 100mL is added simultaneously in chloroform layer After being cleaned, vacuum distillation eliminates chloroform.The triethylamine of 0.5mL is added in the crude product, is purified using silicagel column, Thus 6.27g (yield 74%) white solid is obtained.
1H NMR(400MHz,CDCl3), σ 0.101 (6H, d, J=3.2Hz), σ 0.902 (9H, s), σ 1.368 (9H, t, J =7.6Hz), σ 2.055 (1H, q), σ 2.52-2.58 (1H, dd), σ 3.092 (6H, q, J=7.2Hz), σ 3.874 (2H, t, J= 12.8Hz), σ 4.265 (1H, s), σ 4.864 (1H, t, J=6.8Hz), σ 6.324 (1H, t, J=6.4Hz), σ 7.018 (1H, d, JPH=627Hz), σ 7.424 (1H, br), σ 7.158 (1H, s), σ 8.377 (1H, br)
31P NMR(162MHz,CDCl3),σ2.99
(3) synthetic method of cytidine monomer
[chemical formula 44]
The synthesis of (3-1) 5 '-t-butyldimethylsilyl cytidine
In a nitrogen atmosphere, cytidine 4.86g (20mmol) and pyridine 400mL are added in three-necked flask (500mL), 0 It is stirred at DEG C.The dichloromethane solution 40mL of tertiary butyl chloride dimethylsilane 3.07g (20.4mmol) is slowly added dropwise, in nitrogen To be stirred at room temperature 12 hours under gas atmosphere.Vacuum distillation removal pyridine and methylene chloride, are added water in obtained residue 100mL is extracted twice with chloroform 100mL.Vacuum distillation removal organic layer, is purified using silicagel column, thus obtains 4.93g (yield 69%) white solid.
(3-2) 5 '-t-butyldimethylsilyl -2 '-- H- phosphate synthesis of benzoylcytidine -3 '
In a nitrogen atmosphere, t-butyldimethylsilyl is added in three-necked flask (300mL) and protects cytidine 3.57g (10mmol) and pyridine 100mL, is stirred at -40 DEG C.It was slowly added dropwise chlorobenzoyl chloride 2.11g's (15mmol) with 30 minutes Dichloromethane solution 10mL is stirred 3 hours with -40 DEG C in a nitrogen atmosphere.Dimethylamine hydrochloride 489mg is added in reaction solution (6mmol) dismantles cooling bath and temperature is increased to room temperature by -40 DEG C, stir 2 hours after adding dimethylamine hydrochloride.20 The pyridine solution 20mL of diphenylphosphite 7.03g (30mmol) is slowly added dropwise at DEG C, in a nitrogen atmosphere to be stirred at room temperature 3 Hour.Triethylamine 10mL and water 10mL is added, is stirred at room temperature 1 hour.Vacuum distillation removal pyridine, methylene chloride, To residue in be added water 100mL, be extracted twice with chloroform 100mL.Triethylamine 4mL and water 100mL is added simultaneously in organic layer It is cleaned.Vacuum distillation removal chloroform, the triethylamine of 0.5mL is added in the crude product, is purified using silicagel column, thus Obtain 4.32g (yield 69%) white solid.
1H NMR(400MHz,CDCl3), σ 0.167 (6H, d, J=9.2Hz), σ 0.958 (9H, s), σ 1.241 (9H, t, J =7.2Hz), σ 2.955 (6H, q, J=7.2Hz), σ 3.963 (2H, s), σ 4.544 (1H, s), σ 4.97-5.02 (1H, m), σ 5.411 (1H, t, J=5.6Hz), σ 5.713 (1H, d, J=8.0Hz), σ 6.480 (1H, d, J=6.8Hz), σ 6.897 (1H, d, JPH=628Hz), σ 7.416 (2H, t, J=7.2Hz), σ 7.551 (1H, t, J=7.2Hz), σ 8.005 (1H, d, J=8.0Hz), σ 8.058 (2H, d, J=8.0Hz)
31P NMR(162MHz,CDCl3),σ4.05
(4) synthesis of guanosine monomer
The synthesis of (4-1) N, N- dimethyl carbonamidine protected birds glycosides
[chemical formula 45]
In a nitrogen atmosphere, guanosine 5.66g (20mmol) and methanol 200mL are added in three-necked flask (300mL), in room It is stirred under temperature.The mixed solution of dimethylformamide dimethyl acetal 20mL and methanol 20mL is added, in a nitrogen atmosphere To be stirred at room temperature 16 hours.It filters the solid generated and is cleaned with methanol, be dried under reduced pressure, thus obtain 6.36g (yield 94%) white solid.
The synthesis of (4-2) 5 '-t-butyldimethylsilyl N, N- dimethyl carbonamidine protected birds glycosides
[chemical formula 46]
In a nitrogen atmosphere, N, N- dimethyl carbonamidine protected birds glycosides 3.38g are added in three-necked flask (100mL) (10mmol), dry pyridine 40mL, is stirred at 0 DEG C.Tertiary butyl chloride dimethylsilane 2.26g (15mmol) is slowly added dropwise Dry pyridine solution 20mL, in a nitrogen atmosphere to be stirred at room temperature 24 hours.Vacuum distillation removal pyridine, in obtained residue Middle addition chloroform 100mL, methanol 50mL, water 100mL are simultaneously extracted.After vacuum distillation removal chloroform, methanol, silicagel column is utilized It is purified, thus obtains 4.11g (yield 91%) white solid.
(4-3) 5 '-t-butyldimethylsilyl N, N- dimethyl carbonamidine protection -2- benzoyl guanosine -3 '-H- Phosphate synthesis
[chemical formula 47]
Guanosine 4.53g (10mmol) and dry pyridine through protecting are added in the three-necked flask for being equipped with calcium chloride tube 60mL is stirred at -40 DEG C.The dehydration dichloromethane solution of chlorobenzoyl chloride 2.11g (15mmol) was added dropwise with 30 minutes 20mL is stirred 30 minutes at -40 DEG C.The dry pyridine that diphenylphosphite 7.03g (30mmol) was added dropwise with 15 minutes is molten Liquid 20mL dismantles cooling bath and is impregnated in ice water, stirs 1 hour at 0 DEG C after completion of dropwise addition.Triethylamine 10mL and water is added 10mL is stirred at 0 DEG C and stops reaction.Vacuum distillation removal pyridine, methylene chloride, are added in obtained residue Water is extracted twice with chloroform.Triethylamine and water are added in chloroform layer and is cleaned and eliminates phosphorous acid and phenols.Distillation Chloroform is removed, is dried under reduced pressure and target product is slightly purified using silicagel column after removing triethylamine as far as possible.In turn, will Obtained crude product 5g is dissolved in methanol/chloroform mixed solvent, cleans organic layer with 5wt% aqueous citric acid solution, eliminates The by-product of chloroform layer.Water layer is cleaned with chloroform again and removes by-product, and triethylamine then is added in water layer and alkali is made Property, target product is extracted with chloroform.Distillation removal chloroform, be dried under reduced pressure and remove as far as possible utilize after triethylamine silicagel column into Row purifying, obtains 5.70g (yield 79%) white solid.
1H NMR(400MHz,CDCl3), σ 0.121 (6H, d, J=4.8Hz), σ 0.938 (9H, s), σ 1.234 (9H, t, J =7.2Hz), σ 2.964 (6H, q, J=7.2Hz), σ 3.077 (3H, s), σ 3.233 (3H, s), σ 3.482 (3H, s), σ 3.952 (2H, q, J=10.0Hz), σ 4.478 (1H, s), σ 5.166 (1H, q, J=4.8Hz), σ 5.932 (1H, t, J=5.2Hz), σ 6.330 (1H, d, J=5.6Hz), σ 6.904 (1H, d, JPH=624Hz), σ 7.392 (2H, t, J=7.6Hz), σ 7.539 (1H, T, J=7.6Hz), σ 7.959 (1H, s), σ 8.040 (2H, d, J=7.2Hz), σ 8.725 (1H, br), σ 8.763 (1H, s)
31P NMR(162MHz,CDCl3),σ3.51
The synthesis of (embodiment 2) RNA oligomer
Using the monomer of above-mentioned synthesis, RNA oligomer has been synthesized by liquid phase synthesis.
[table 2]
Table 2
Extension is carried out under above-mentioned reaction condition, and ether precipitating is carried out in each extension, is thus purified.
[table 3]
Composite result when 3.20 base of table extends
Characterization
Obtain extending to 5 '-TBDMS-rU of the 20th base19After dT-3 '-O-suc-O-PEG, 2 ' positions are carried out with ethylenediamine Benzoyl protection and 3 '-suc-O-PEG deprotection, then carry out HPLC purifying, thus obtain 5 '-TBDMS-rU19dT- 3'-OH.In turn, the deprotection of 5 ' position TBDMS bases is carried out by TBAF, and LC- is carried out to the sample for having carried out HPLC purifying again MS analysis, has thereby confirmed that and 5 '-HO-rU19The consistent m/z=6058.90 of the molecular weight of dT-3 '-OH (theoretical value: 6059.38) (referring to Fig.1).
The extension of various bases is imported
[table 4]
Reaction condition when 4.10 base of table extends
The result that 10 bases are extended to when importing each base kind is shown in following table 5.
[table 5]
Composite result when 5.10 base of table extends
In the same manner as the case where extending to 20 base, obtain 5 '-TBDMS-rU3rCrGrArU3dT-3’-O-suc-O-PEG Afterwards, the deprotection of the benzoyl protection and 3 '-suc-O-PEG of 2 ' positions is carried out with ethylenediamine.HPLC purifying is carried out, obtains 5 '- TBDMS-rU3rCrGrArU3dT-3'-OH.In turn, the deprotection of 5 ' position TBDMS bases is carried out by TBAF, again to progress The sample of HPLC purifying carries out LC-MS analysis, has thereby confirmed that and 5 '-HO-rU3rCrGrArU3The molecular weight of dT-3 '-OH is consistent M/z=3058.60 (theoretical value: 3058.82) (referring to Fig. 2).
As described above, the present invention is illustrated using the preferred embodiments of the present invention, it should be understood that the present invention is only Its range is explained based on claims.It is also understood as the patent quoted in this specification, patent application and other documents Its content itself using in a manner of specific record in the same manner as content in this manual as being directed to the reference of this specification extremely In the present invention.
Industrial availability
The present invention is useful in the pharmaceutical industries and reagent industry using nucleic acid drug etc..
Sequence number 1: the sequence of the base number 10 in table 3
Sequence number 2: the sequence of the base number 15 in table 3
Sequence number 3: the sequence of the base number 19 in table 3
Sequence number 4: the sequence of the base number 20 in table 3
Sequence number 5: the sequence of the base number 10 in table 5
Sequence table
<110>gene design Co., Ltd (GeneDesign, Inc.)
State-run research and development legal person industrial technology comprehensive study institute (National Institute of Advanced Industrial Science and Technology)
<120>synthetic method of ribonucleic acid H- phosphate ester monomer and the oligonucleotide synthesis of the monomer has been used
<130> JD003PCT
<150> JP 2016-122554
<151> 2016-06-21
<160> 5
<170> PatentIn version 3.5
<210> 1
<211> 10
<212> DNA/RNA
<213>artificial sequence (artificial sequence)
<220>
<223>DNA/RNA molecule is combined
<220>
<221> RNA
<222> (1)..(9)
<220>
<221> DNA
<222> (10)..(10)
<400> 1
uuuuuuuuut 10
<210> 2
<211> 15
<212> DNA/RNA
<213>artificial sequence (artificial sequence)
<220>
<223>DNA/RNA molecule is combined
<220>
<221> RNA
<222> (1)..(14)
<220>
<221> DNA
<222> (15)..(15)
<400> 2
uuuuuuuuuu uuuut 15
<210> 3
<211> 19
<212> DNA/RNA
<213>artificial sequence (artificial sequence)
<220>
<223>DNA/RNA molecule is combined
<220>
<221> RNA
<222> (1)..(18)
<220>
<221> DNA
<222> (19)..(19)
<400> 3
uuuuuuuuuu uuuuuuuut 19
<210> 4
<211> 20
<212> DNA/RNA
<213>artificial sequence (artificial sequence)
<220>
<223>DNA/RNA molecule is combined
<220>
<221> RNA
<222> (1)..(19)
<220>
<221> DNA
<222> (20)..(20)
<400> 4
uuuuuuuuuu uuuuuuuuut 20
<210> 5
<211> 10
<212> DNA/RNA
<213>artificial sequence (artificial sequence)
<220>
<223>DNA/RNA molecule is combined
<220>
<221> RNA
<222> (1)..(9)
<220>
<221> DNA
<222> (10)..(10)
<400> 5
uuucgauuut 10

Claims (19)

1. a kind of method for manufacturing general formula compound represented below or its salt or its solvate,
[chemical formula 1]
In formula (1), R1Indicate the base selected from the group being made of substituted or unsubstituted aryl and substituted or unsubstituted heteroaryl Group, R2Indicating the protecting group of hydrogen atom or hydroxyl, B indicates nucleic acid base optionally with protecting group,
This method includes process 1 and process 2,
Process 1 is that [chemical formula 2] is made to react the process for obtaining [chemical formula 4] with [chemical formula 3],
[chemical formula 2]
[chemical formula 3]
In formula (3), X is halogen atom,
[chemical formula 4]
Process 2 is to make [chemical formula 5] and P (OR3)(OR3’) OH react obtain [chemical formula 6] process,
[chemical formula 5]
[chemical formula 6]
Formula P (OR3)(OR3’) in OH, R3And R3’It separately indicates selected from by substituted or unsubstituted aryl and substitution or not The group of the group of substituted heteroaryl composition,
The process 1 and the process 2 carry out in 1 reaction vessel.
2. according to the method described in claim 1, wherein, the optionally nucleic acid base with protecting group is selected from by forming as follows Group,
[chemical formula 7]
In formula (7), R3a、R3bAnd R3cIt separately indicates selected from by hydrogen atom, substituted or unsubstituted linear or branched alkyl group Acyl group, substituted or unsubstituted aryl-acyl, substituted or unsubstituted alkyl, substituted or unsubstituted naphthenic base, substitution or not The group or R of the group of substituted aryl and substituted or unsubstituted heteroaryl composition3aWith R3bIt is formed together amidine type protecting group Group.
3. according to the method described in claim 2, wherein, the amidine type protecting group is N, N- dimethylformamidinyl or N, N- diformazan Base ethanamidine base.
4. method described in any one of claim 1 to 3, wherein R1For substituted or unsubstituted aryl.
5. according to the method described in claim 4, wherein, the substituted or unsubstituted aryl is phenyl.
6. method according to any one of claims 1 to 5, wherein the protecting group of the hydroxyl is selected from being protected by ether system Protect the protecting group of the group of base, silyl ether system protecting group, acetal system protecting group and acyl group system protecting group composition.
7. according to the method described in claim 6, wherein, ether system protecting group are as follows:
[chemical formula 8]
In formula (R2a), R4a1、R4a2、R4a3、R4a4、R4a5、R4b1、R4b2、R4b3、R4b4、R4b5、R4c1、R4c2、R4c3、R4c4And R4c5Point It independently is not selected from by hydrogen atom, substituted or unsubstituted straight or branched alkoxyl and substituted or unsubstituted straight chain or branch The group of the group of alkyl group composition.
8. according to the method described in claim 7, wherein, the substituted or unsubstituted straight or branched alkoxyl be straight chain or Branch perfluoro alkoxy.
9. according to the method described in claim 7, wherein, the substituted or unsubstituted linear or branched alkyl group is straight chain or branch Chain perfluoroalkyl.
10. according to the method described in claim 6, wherein, silyl ether system protecting group are as follows:
[chemical formula 9]
In formula (R2b), R4d、R4eAnd R4fSeparately for selected from by substituted or unsubstituted linear or branched alkyl group, substitution or The group of the group of unsubstituted aryl and substituted or unsubstituted alkenyl composition.
11. according to the method described in claim 10, wherein, the substituted or unsubstituted alkenyl is allyl.
12. according to the method described in claim 6, wherein, silyl ether system protecting group are as follows:
[chemical formula 10]
In formula (R2c), R4g、R4hAnd R4iSeparately for selected from by substituted or unsubstituted alkyl, substituted or unsubstituted first The group of the group of silylation and substituted or unsubstituted silicyl oxygroup alkyl composition.
13. according to the method for claim 12, wherein the silicyl is replaced by 1~3 alkyl.
14. according to the method for claim 12, wherein the substituted or unsubstituted alkyl is selected from by replacing or not taking The linear or branched alkyl group in generation, substituted or unsubstituted linear chain or branched chain alkenyl, substituted or unsubstituted linear chain or branched chain alkynyl, It is substituted or unsubstituted naphthenic base, substituted or unsubstituted cycloalkenyl, substituted or unsubstituted aryl, substituted or unsubstituted straight The group of the group of chain or branch aryl alkyl composition.
15. according to the method for claim 14, wherein the substituted or unsubstituted linear or branched alkyl group be straight chain or Branched haloalkyl.
16. according to the method for claim 15, wherein the linear chain or branched chain halogenated alkyl is linear chain or branched chain perfluor alkane Base.
17. a kind of for manufacturing the kit of following general formula compound represented, wherein the general formula are as follows:
[chemical formula 13]
Wherein, in formula (13), each mark is identical as aforementioned meaning,
The kit includes:
[chemical formula 11]
[chemical formula 12]
With
P(OR3)(OR3’) OH,
In formula (11), R2Indicating the protecting group of hydrogen atom or hydroxyl, B indicates nucleic acid base optionally with protecting group,
In formula (12), R1Indicate the base selected from the group being made of substituted or unsubstituted aryl and substituted or unsubstituted heteroaryl Group, X is halogen atom,
Formula P (OR3)(OR3’) in OH, R3And R3’It separately indicates selected from by substituted or unsubstituted aryl and substitution or not The group of the group of substituted heteroaryl composition.
18. it is a kind of for selectively manufacturing the phosphorus reagent of following general formula compound represented or its salt or its solvate, In, the general formula are as follows:
[chemical formula 14]
In formula (14), R1Indicate the base selected from the group being made of substituted or unsubstituted aryl and substituted or unsubstituted heteroaryl Group, R2Indicating the protecting group of hydrogen atom or hydroxyl, B indicates nucleic acid base optionally with protecting group,
The phosphorus reagent includes P (OR3)(OR3’) OH,
In above formula, R3And R3’It separately indicates selected from by substituted or unsubstituted aryl and substituted or unsubstituted heteroaryl The group of the group of composition.
19. a kind of P (OR3)(OR3’) OH is for selectively manufacturing following general formula compound represented or its salt or its solvent Application in compound, wherein the general formula are as follows:
[chemical formula 15]
In formula (15), R1Indicate the base selected from the group being made of substituted or unsubstituted aryl and substituted or unsubstituted heteroaryl Group, R2Indicating the protecting group of hydrogen atom or hydroxyl, B indicates nucleic acid base optionally with protecting group,
Formula P (OR3)(OR3’) in OH, R3And R3’It separately indicates selected from by substituted or unsubstituted aryl and substitution or not The group of the group of substituted heteroaryl composition.
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